Turnip Yellow Mosaic Virus 3’UTR as a translational enhancer in Saccharomyces cerevisiae

Lisa BauerMicrobiology

Mentors: Daiki MatsudaDr. Theo Dreher

Background

Turnip Yellow Mosaic Virus (TYMV) Single-stranded positive-sense RNA virus

p69

p206

XmnIDraI

3 overlapping reading frames (ORFs):-p69: Overlapping Protein-p206: Replication Protein-Coat Protein (CP)

CP

5’UTR3’UTR

TLS

3’ tRNA-like structure

UAAU

G-CC-GG-CA-UG-CGU-AC-GU-AG-CU-AC-G

AC

UA

CC

AA

C U

C GU

CCCG

GGGC

CCC

GGG

CUCU

UCGGA A

AGCCU

UCAU G

3´

GAUUC-GU-AG-C

UUU

AAA

CA-U

UCUUGAAU C

CC AC

Major valine identity nts in the anticodon loop

-Val

Enhanced translation with 3’UTR seen in plant cells (Matsuda et al., 2002)

eIF4E

A

AC

C

C

C A C

Saccharomyces cerevisiae

Fungi Eukaryotic Unicellular

Why is yeast ideal? Small genome Entire genome known Genetic system with characterized

mutants Simple system to use

Goal

The primary goal was to simulate the same translational phenomenon seen in plant cells of pre-existing RNA constructs in yeast cellsUsed pre-existing RNA constructs from

Daiki Matsuda and Wei Wei Chiu

Methodology of Yeast Electroporation

Gallie et al. (1992) Development of yeast electroporation

system for expressing luciferase proteinCap and Poly A tail essential for efficient

translationSearfoss et al. (2004)

Yeast electroporation method used

Experimental Procedure

Preparation of yeast spheroplasts Strain BY4741

98 mins doubling time in YEPD mediumGrow to 0.6 OD

Suspend in Buffer A (Sorbitol, TrisCl, MgCl2, DTT, ß-

mercaptoethanol)Lyticase treatment

BY4741 (18 mins)90 minute recovery

Experimental Process

2. In vitro run-off transcription by T7 RNA polymerase

(with/ without cap analog)*

1. Linearize plasmid

3. RNA transfection

5. Cell lysis

6. Luciferase reaction

LUC

Protoplasts of cowpea leaves

S. cerevisiae spheroplasts

vs.4. Translation at RT

*Daiki Matsuda

RNA constructs

Cap GLG-pACap +Tail +

Controls:

Cap GLGGLG-pAGLG

TY 3’ UTR:

Cap vec-L-TYsgCap vec-L-Bamvec-L-TYsgvec-L-Bam

TY3’gTY3’sgTY3’sg(CGC)TY3’sg(GAC)TY3’BamTY3’PvuTY3’Dra genomic

subgenomic

Thanks to Wei Wei Chiu and Daiki Matsuda for use of constructs

Poly A & Cap Effects

Cap GLG-pA

GLG-pA

Cap GLG

GLG

1

22.2

0 5 10 15 20 25

Light Units (x108)

Poly A EffectCap Effect

1

68.3

1

27.0

1

83.0

3’ UTR & Cap Effects

Cap vec-L-Bam

vec-L-Bam

vec-L-TYsg

Cap vec-L-TYsg

0 5 10 15 20Light Units (x109)

1

5.29

1

33.55

TY 3’ and Cap Synergy: 33.55/5.29= 6.34 20.3/3.31= 6.12

July 27 August 6

1

3.31

1

20.3

0 5 10 15 20Light Units (x109)

Synergy in plant cells: ~10

3’UTR Effects

August 17 August 20

TY3’Dra

TY3’Bam

TY3’Pvu

TY3’g

TY3’sg

0 2 4 6 8 10 12

Light Units (x109)

2.10

1

0.09

0.24

0.08

2.24

1

0.07

0.2

0.07

0 2 4 6 8 10 12

Light Units (x109)

Plant Cell Data

Yeast Cell Data

2.18

1

0.21

0.13

0.09

UAAU

G-CC-GG-CA-UG-CGU-AC-GU-AG-CU-AC-G

AC

UA

CC

AA

C U

C GU

CCCG

GGGC

CCC

GGG

CUCU

UCGGA A

AGCCU

UCAU G

3´

GAUUC-GU-AG-C

UUU

AAA

CA-U

UCUUGAAU C

CC AC -Val

A

AC

CC

TY3’ Valylation Effect

TY3’sg(CGC)

TY3’sg

TY3’sg(GAC)

Plant cell dataExperiment 1

Experiment 2

0 2 4 6 8 10 12

Light Units (x109)

GG

1

0.46

0.49

0.61

0.6

0.14

0.8

0.6

0.8

0.46

0.49

1

0.49

0.46

C

Conclusions

3’TYMV:30 fold 3’ effect; similar to poly A effect~27 fold TY cap effect3’ TY synergy with cap ~ 6 fold

3’UTR:Subgenomic 2x genomic 3’endDra and Bam cuts both ~7% of wild type;

TLS important factorNon-valylation less effect than expectedNon-valylation less effect than expected

Next Steps

Electroporation with W303 strain Utilize mutant yeast strains

RNA turnover Initiation factor mutants

0.0E+00

2.0E+08

4.0E+08

6.0E+08

8.0E+08

1.0E+09

1.2E+09

1.4E+09

0 20 40 60 80 100

Time (mins)

Lig

ht

Un

its

TY 3'sg

TY 3' CGC

TY Bam

GAC(wobble)

TY 3'g

TY 3'Dra1

TY Pvu

Acknowledgements

Dr. Theo Dreher Daiki Matsuda Kevin Ahern Howard Hughes Medical

Institute National Science

Foundation